The establishment of the thymic microenvironment during development and early in life is crucial to enable the production of functional T cells. Conversely, thymic degeneration (involution) in aging organisms results in decreased T cell output, increased susceptibility to opportunistic infection, auto-immunity, cancer, and a decreased capacity to respond to vaccination. In addition, thymic involution prevents proper restoration of immune function after chemotherapy, ionizing radiation exposure, and infection. Our long-term objective is to gain a better understanding of the mechanisms underlying the deterioration of the thymic microenvironment during aging, focusing on the biology of thymic epithelial cells (TECs), which provide a microenvironment conducive to T cell expansion and differentiation. Little is known about the mechanisms that regulate thymus involution during aging and in response to stress, but we have made observations that indicate that certain manipulations of the thymic stroma can prevent involution. In particular, our most recent studies and accumulating evidence in many cell types during aging suggest that thymic involution is accompanied and caused by changes in the chromatin structure of TECs. These epigenetic changes may not only control normal involution but also may hamper therapeutic attempts to regenerate thymic function during aging. In the thymus, TECs are largely outnumbered by T cells. A major challenge in studies of the thymic epithelium is the low number of cells that can be harvested per mouse, which has prevented genomic analyses of these populations thus far. We will use two approaches to query changes in chromatin structure (ATAC- seq) and DNA methylation (HELP-GT) that occur during aging in TEC populations in mice. These two approaches can be performed with low number of cells (thousands, instead of millions). Combined with gene expression analyses (RNA-seq), these experiments will provide the first detailed report of gene expression and chromatin structure changes in the aging thymic epithelium. Performing these studies in control mice and mice mutant for pathways involved in thymic involution (e.g. the Rb pathway and the Foxn1 transcription factor) will help elucidate molecular mechanisms of thymic involution. Better knowledge of the mechanisms controlling gene expression in TECs may ultimately lead to novel means to control the thymic microenvironment and boost immune function in a wide range of aging patients.